Genetic Mapping And Gene Dissection Of Nutritional And Other Important Economic Traits Of The Oyster

Oysters are known as “The Milk of the sea”,they are rich in nutrition,such as glycogen,glutamate,taurine,zinc,selenium,EPA,and DHA.Various nutritional products of the oyster further verified its nutritional value.However,problems like germplasm degradation and unreasonable farming mode,had been a barrier,and resulted in the low profits of the oyster industry.Because of which,breeding process of oysters towards fast-growing,high condition index,and high nutrtion,should be paid more attention.Apart from growth and nutritional traits,summer mortality also limited the development of the oyster industry.Because of which,apoptosis caused wide attention as its role in immune defense,homeostasis,etc..As an initiator caspase,caspase-8 plays a key role in the extrinsic apoptotic pathway of the vertebrate.For the invertebrate,the apoptotic pathway varied with the species.The problem that whether extrinsic apoptotic pathway is conserved in Crassostrea gigas(C.gigas)and what's the role of caspase-8 in C.gigas needs further study.Crassostrea gigas,also named the Pacific oyster,is a vital economic shellfish that cultured all over the world,and meantime the major cultivated shellfish of Huanghai and Bohai Sea of China.In this study,we take growth and nutritional traits,as well as caspase-8 gene of C.gigas as research object.On one hand,we constructed specific population and family,and detected the association between molecular markers and traits by genome-wide association study(GWAS)and linkage analysis.Detected traits include growth and nutritional traits,the latter includes glycogen,amino acids,and fatty acids.Our purpose is providing candidate loci and causative genes for molecular breeding,and laying the foundation for the genetic parsing of growth and nutritional traits in C.gigas.On the other hand,we studied the function of caspase-8 gene in C.gigas.We detected its interaction with upper protein in the pathway,analyzed its expression pattern among different tissues and developmental stages,and towards bacteria and virus challenge.This work will help explain the apoptosis and immune function of caspase-8 in C.gigas,and provide basis for the studing of apoptotic process of the invertebrate.The main contents and conclusions are as follows:1.Genetic parsing of growth and nutritional traits of the oyster.(1)Genome-wide association study of growth and nutritional traits.We constructed 427 sub-families by hybridzing the maternal oysters collected from five countries and 23 locations with the same paternal oyster of Qingdao,Shandong Province.All the families were cultivated in the same environment in Qingdao.We genotyped all the maternal oysters by whole-genome-resequencing,and detected the average phenotype values of the F1 family to represent the maternal oyster.Finally,we identified 33 significant positive signals of 20 traits,and found some causative genes that were associated with the traits by gene annotation of LD(Linkage disequilibrium)interval.Candidate genes associated with fatty acid traits,like NFYA and CYP7A1,can directly participate in the fatty acid metabolism and regulation,and show great value of molecular breeding.(2)Genetic map construction and quantitative triat loci(QTL)mapping of growth and nutritional traits.We randomly selected 169 individuals from the full-family constructed by hybridzing maternal oyster from Changli and paternal oyster from Qingdao.Using double-digest genotyping by sequencing(dd GBS)method,we constructed a high-density genetic map.The consensus map contained 5024 evenly distributed markers on ten linkage groups(LG).The map length was 1982.07 c M,with an average distinct marker interval of 0.68 c M,thus representing the densest genetic map produced for C.gigas.According to the high collinearity between the consensus map and the oyster genome,1574 scaffold(about 70%)of the genome of C.gigas was first anchored to the LGs of the consensus map.Using this map,we also conducted QTL analysis for growth and nutritional traits.Overall,41 QTLs were detected for 17 traits.Candidate genes identified in the QTL interval include both genes related to growth traits and genes participating in amino acid and fatty acid metabolism and regulation.Of which,six candidate genes could influence phenotype by regulating m RNA expression levels,and thus are key targets for molecular breeding of growth and nutritional traits.These six genes include AMY?BMP1?P5CS?GR?ADAMTS,and DYRK.In summary,both GWAS and QTL mapping for growth and nutritional traits provided lots of valuable markers and candidate genes for molecular assisted selection(MAS).Combined with gene function studying method,genetic mechanism underlying related traits will be clarified.Besides,it's the first time to perform genetic parsing of amino acid and fatty acid traits in C.gigas,despite its high nutritional value.Our work can directly provide benefits for the oyster industry.Candidate genes both identified in fatty acid peak SNP linkage interval by GWAS and identified by linkage analysis that can influence phenotype by changing m RNA expression levels are key objects of molecular breeding of important economic traits of the oyster,and will promote the process of molecular breeding.2.Functional characterization of CgCaspase8-2.In this study,we cloned the first caspase-8-like gene(CgCaspase8-2)of C.gigas.The deduced Cg Caspase8-2 protein contained typical domains and the conserved pentapeptide motif ‘QACQG' of caspase-8.Phylogenetic analysis indicated that Cg Caspase8-2 was clustered with initiator caspases in the invertebrate subgroup,but the similarity between Cg Caspase8-2 and other invertebrate caspase-8s was low.Cg Caspase8-2 protein was localized in the cytoplasm,and over-expression of Cg Caspase8-2 in HEK293 T cells induced cell death,suggesting a role in apoptosis.However,Cg Caspase8-2 didn't combine with the upper adapter protein(Cg FADD)of the pathway,so we suggested it didn't take part in the extrinsic apoptotic pathway.Quantitative real-time PCR results demonstrated that Cg Caspase8-2 was highly expressed in the hemolymph and gill,suggesting its underlying immune function.Furthermore,Cg Caspase8-2 gene expression analysis in response to bacterial and poly(I:C)challenge showed that Cg Caspase8-2 was specifically involved in immune response against virus rather than bacteria.